The access of drugs into the central nervous system (CNS) is regulated by the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB). A large body of evidence supports perturbation of these barriers in neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. Modifications to the BBB and BSCB are also reported in amyotrophic lateral sclerosis (ALS), albeit these modifications have received less attention relative to those in other neurodegenerative diseases. Such alterations to the BBB and BSCB have the potential to impact on CNS exposure of drugs in ALS, modulating the effectiveness of drugs intended to reach the brain and the toxicity of drugs that are not intended to reach the brain. Given the clinical importance of these phenomena, this review will summarise reported modifications to the BBB and BSCB in ALS, discuss their impact on CNS drug exposure and suggest further research directions so as to optimise medicine use in people with ALS.
Background and Purpose Refractory status epilepticus is a clinical emergency associated with high mortality and morbidity. Increasing evidence suggests neuroinflammatory pathways contribute to the development of drug-refractoriness during status epilepticus. The ATP-gated P2X7 receptor (P2X7R) has been described as potential link between inflammation and increased hyperexcitability. The aim of the present study was to determine the contribution of the P2X7R to drug-refractory status epilepticus and its therapeutic potential. Experimental Approach Status epilepticus was induced via a unilateral microinjection of kainic acid into the amygdala in adult mice. Severity of status epilepticus was compared in animals overexpressing or knock-out in the P2X7R, after inflammatory priming by the pre-injection of bacterial lipopolysaccharide (LPS) and in mice treated with P2X7R-targeting and anti-inflammatory drugs. Key Results P2X7R overexpressing mice were unresponsive to several anticonvulsants (lorazepam, midazolam, phenytoin and carbamazepine) during status epilepticus. P2X7R expression was increased in microglia during drug-refractory status epilepticus, P2X7R overexpression led to a pro-inflammatory phenotype in microglia during status epilepticus and the anti-inflammatory drug minocycline restored normal responsiveness to anticonvulsants in P2X7R overexpressing mice. Pre-treatment of wildtype mice with LPS increased P2X7R levels in the brain and promoted the development of pharmaco-resistant status epilepticus, which was overcome by either a genetic deletion of the P2X7R or the administration of the P2X7R antagonists AFC-5128 or ITH15004. Conclusion and Implications Our results demonstrate that P2X7R-induced pro-inflammatory effects contribute to resistance to pharmacotherapy during status epilepticus and suggest therapies targeting the P2X7R as novel adjunctive treatments for drug-refractory status epilepticus.
In the retina, mineralocorticoid receptor (MR), expressed in vessels, glial and neuronal cells, is mainly activated by glucocorticoids. Under pathological conditions, ocular MR expression and corticoids change, leading in most cases to MR overactivation. Experimental models using MR agonists or antagonists, administered systemically or intraocularly, acutely or chronically and transgenic models, allowed to identify the deleterious consequences of MR pathway overactivation. Among them, oxidative stress, inflammation, deregulation of hydro-ionic channels, alteration of choroidal vasculature, angiogenesis and cell death, are common to major retinal diseases. Specific MR antagonists showed efficacy in models of diabetic retinopathy, ischaemia, retinal and choroidal angiogenesis and in models of glaucoma. It is highly likely that MR antagonists will find a place in the therapeutic arsenal of age-related macular degeneration, diabetic retinopathy, glaucoma and in pachychoroid associated diseases. Their use in humans is still limited by the need of biomarkers of MR activation and specific ocular formulations.
G protein-coupled receptors modulate a plethora of physiological processes and mediate the effects of one-third of FDA-approved drugs. Notably, depending on which ligand has activated a particular receptor, it can engage different intracellular transducers. This paradigm of ligand-dependent ‘biased signaling’ dictates a need to advance beyond the level of receptors to consider the combined ligand-receptor pair in order to understand physiological signaling. Bias signaling also has the potential to improve medicines by reducing adverse effects. However, this is challenged by inconsistent interpretation of results and lack of commonly agreed guidelines. Here, we present recommended terminology and guidelines to conduct, report and quantify bias in a comparable and reproducible fashion. We expect these recommendations will facilitate a common understanding of experiments and findings across basic receptor research and drug discovery, while the area and the analytical methods to measure bias are still evolving, especially in complex cellular, tissue and organismal systems.
Background and Purpose: Despite availability of a variety of treatment options, many asthma patients have poorly controlled disease with frequent exacerbations. Proteinase-activated receptor-2 (PAR2) has been identified in pre-clinical animal models as important to asthma initiation and progression following allergen exposure. Proteinase activation of PAR2 induces intracellular Ca2+, mitogen activated protein kinase (MAPK) and -arrestin signaling the airway, leading to both inflammatory and protective effects. We have developed C391, a potent PAR2 antagonist effective in blocking peptidomimetic- and trypsin-induced PAR2 signaling in vitro as well as reducing inflammatory PAR2-associated pain in vivo. We hypothesized that PAR2 reduction with C391 would attenuate allergen-induced asthma indicators in murine models. Experimental Approach: We evaluated the ability for C391 to alter Alternaria alternata-induced PAR2 signaling pathways in vitro using a human airway epithelial cell line that naturally expresses PAR2 (16HBE14o-) and a transfected embryonic cell line (HEK 293). We next evaluated the ability for C391 to reduce A. alternata-induced asthma indicators in vivo in two murine strains. Key Results: C391 blocked A. alternata-induced, PAR2-dependent Ca2+ and MAPK signaling in 16HBE14o- cells, as well as -arrestin recruitment in HEK 293 cells. C391 effectively attenuated A. alternata-induced inflammation, mucus production, mucus cell hyperplasia and airway hyperresponsiveness in acute asthma murine models. Conclusions and Implications: To our knowledge, this is the first demonstration of pharmacological intervention of PAR2 to reduce allergen-induced asthma indicators in vivo. These data support further development of PAR2 antagonists as potential first-in-class allergic asthma drugs.
Peptides play a key role in controlling many physiological and neurobiological pathways. Many bioactive peptides require a C-terminal α-amide for full activity. The bifunctional enzyme catalyzing α-amidation, peptidylglycine α-amidating monooxygenase (PAM), is the sole enzyme responsible for amidated peptide biosynthesis, from Chlamydomonas reinhardtii to Homo sapiens. Many neuronal and endocrine functions are dependent upon amidated peptides; additional amidated peptides are growth promoters in tumors. The amidation reaction occurs in two steps, glycine α-hydroxylation followed by dealkylation to generate the α-amide product. Currently, most potentially useful inhibitors target the first reaction, which is rate-limiting. PAM is a membrane-bound enzyme that visits the cell surface during peptide secretion. PAM is then used again in the biosynthetic pathway, meaning that cell-impermeable inhibitors or inactivators could have therapeutic value for the treatment of cancer or psychiatric abnormalities. To date, inhibitor design has not fully exploited the structures and mechanistic details of PAM.
Post-operative ileus (POI) is a frequent complication after abdominal surgery. The consequences of POI can be potentially serious such as bronchial inhalation or acute functional renal failure. Numerous advances in peri-operative management, particularly early rehabilitation, have made it possible to decrease POI. Despite this, the rate of prolonged POI ileus remains high and can be as high as 25% of patients in colorectal surgery. From a pathophysiological point of view, POI has two phases, an early neurological phase and a later inflammatory phase, to which we could add a “pharmacological” phase during which analgesic drugs, particularly opiates, play a central role. The aim of this review article is to describe the phases of the pathophysiology of POI, to analyse the pharmacological treatments currently available through published clinical trials and finally to discuss the different research areas for potential pharmacological targets.
Aldosterone binds to the mineralocorticoid receptor (MR), a transcription factor of the nuclear receptor family, present in the kidney and in various other non-epithelial cells including the heart and the vasculature (Cannavo et al., 2018). Indeed, extra-renal pathophysiological effects of this hormone have been characterized, extending its actions to the cardiovascular (CV) system (Messaoudi et al., 2012). A growing body of clinical and pre-clinical evidence suggests that MR activation plays an important pathophysiological role in CV remodeling by promoting cardiac hypertrophy, fibrosis, arterial stiffness, as well as in inflammation and oxidative stress (Bauersachs et al., 2015). The following review article outlines the role of MR in CV disease with a focus on myocardial remodeling and heart failure (HF) including clinical trials as well as cellular and animal studies.
Background and purpose. Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) recently identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and get insight into the MQ1 molecular mode of action. Experimental approach. We used a bio-guided screening assay to identify novel MQs and placed them phylogenetically. Several newly identified MQs were produced by solid phase peptide synthesis. They were characterized in vitro by binding and functional tests andin vivo by diuresis measurement in rats. Key results. Eight additional MQs were identified with nanomolar affinities for the V2R, all antagonists. MQs form a new subgroup in the Kunitz family, close to the V2R non-active dendrotoxins and to 2 V2R active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted 5 specific V2R positions. Four of them are involved in V2R activity and belong to the 2 large MQ1 loops. We finally determined that 8 positions, part of these 2 loops, interact with the V2R. The variant MQ1-K39A showed specificity for the human versus the rat V2R . Conclusions and implications. A third function and mode of action is now associated with the Kunitz-peptides. The number of MQ1 residues involved in V2R binding is large and may explain its absolute selectivity. MQ1-K39A represents the first step in the improvement of the MQ1 design for medicinal perspective.
Fungal infections cause serious problems in many aspects of human life; especially infections by fungal species represent problems in immunocompromised patients. Current antifungal antibiotics target various metabolic pathways, predominantly the cell wall or cellular membrane. However, numerous compounds are available to combat fungal infections, their efficacy is far from being satisfactory and some of them display substantial toxicity. The emerging resistance represents a serious issue as well; thus, there is a considerable need for new anti-fungal compounds with lower toxicity and higher effectiveness. One of the unique antifungal antibiotics is sordarin, the only known compound that acts on the fungal translational machinery per se. It has been shown that sordarin inhibits protein synthesis at the elongation step of the translational cycle, acting on eukaryotic elongation-factor-2. In this review, we are aiming to deliver a robust scientific platform promoting the development of antifungal compounds, especially focusing on molecular action of sordarin.
Liver fibrosis induced by chronic hepatic injury remains as a major cause of morbidity and mortality worldwide. Identification of susceptibility/prognosis factors and new therapeutic tools for treating hepatic fibrotic disorders of various etiologies are urgent medical needs. Cortistatin is a neuropeptide with potent anti-inflammatory and anti-fibrotic activities in lung that binds to receptors that are expressed in liver fibroblasts and hepatic stellate cells. Here, we evaluated the capacity of cortistatin to regulate liver fibrosis. We initially found that hepatic expression of cortistatin inversely correlated with liver fibrosis grade in mice and humans with hepatic disorders. Cortistatin-deficient mice showed exacerbated signs of liver damage and fibrosis and increased mortality rates when challenged to hepatotoxic and cholestatic injury. Compared to wild-type mice, non-parenchymal liver cells isolated from cortistatin-deficient mice showed increased presence of cells with activated myofibroblast phenotypes and a differential genetic signature that is indicative of activated hepatic stellate cells and periportal fibroblasts and of myofibroblasts with active contractile apparatus. Cortistatin treatment reversed in vivo and in vitro these exaggerated fibrogenic phenotypes and protected from progression to severe liver fibrosis in response to hepatic injury. In conclusion, we identify cortistatin as an endogenous molecular break of liver fibrosis and its deficiency as a potential poor-prognosis marker for chronic hepatic disorders that course with fibrosis. Cortistatin-based therapies emerge as attractive strategies for ameliorating severe hepatic fibrosis.
The insulin receptor is a membrane protein responsible for regulation of nutrient balance and therefore an attractive target in the treatment of diabetes and metabolic syndrome. Pharmacology of the insulin receptor involves two distinct mechanisms, (1) activation of the receptor by insulin mimetics that bind in the extracellular domain and (2) inhibition of the receptor tyrosine kinase enzymatic activity in the cytoplasmic domain. While a complete structural picture of the full-length receptor comprising the entire sequence covering extracellular, transmembrane, juxtamembrane and cytoplasmic domains is still elusive, recent progress through cryoelectron microscopy has made it possible to describe the initial insulin ligand binding events at atomistic detail. We utilize this opportunity to obtain structural insights into the pharmacology of the insulin receptor. To this end, we conducted a comprehensive docking study of known ligands to the new structures of the receptor. Through this approach, we provide an in-depth, structure-based review of human insulin receptor pharmacology in light of the new structures.
The present work analyses in detail the published data on ChAdOx1 nCoV-19 vaccine and provides arguments for the involvement of anti-vector immunity and of SARS-CoV-2 variants on the efficacy of ChAdOx1 nCoV-19 vaccine. First, it is suggested that anti-vector immunity takes place as the regimen of homologous vaccination with ChAdOx1 nCoV-19 vaccine is applied and interferes with efficacy of the vaccine when the interval between prime and boost doses is less than three months. Second, longitudinal studies suggest that ChAdOx1 nCoV-19 vaccine provides sub-optimal efficacy against UK variant of SARS-CoV-2, which appears to have an increased transmissibility over the ancestral SARS-CoV-2 among vaccinated people. At the moment, ChAdOx1 nCoV-19 vaccine is able to reduce the severity of symptoms and transmissibility; however, if the vaccinated individuals do not maintain everyday preventive actions, they could turn into potential spreaders, thus accelerating the process of generation of new viral variants due to the selective pressure of immune response. Prediction and possible consequences of the SARS-CoV-2 evolution and repeated anti-SARS-CoV-2 vaccinations are discussed. Since the impact of emerging SARS-CoV-2 variants suggests that vaccines are unlikely to be effective in quickly solving the pandemic crisis, it is highlighted the need to keep searching for new and more efficacious pharmacotherapy for COVID-19, such as those targeting ACE2 and ADAM17 zinc-metalloprotease activities.
Bruton’s tyrosine kinase (BTK) is a non-receptor kinase best known for its role in B lymphocyte development that is critical for proliferation, and survival of leukaemia cells in B cell malignancies. However, BTK is expressed in myeloid cells, particularly monocytes and macrophages where its inhibition has been reported to exhibit anti-inflammatory properties. Therefore, we explored the role of BTK on the migration of myeloid cells in vitro and in vivo. Using the zymosan induced peritonitis model of sterile inflammation we demonstrated that acute (1 h prior to zymosan) inhibition of BTK using a wide range of FDA (Ibrutinib and Acalabrutinib) and non-FDA approved inhibitors (ONO-4059, CNX-774, Olumatinib and LFM-A13) reduced neutrophil and monocyte recruitment. XID mice, which have a point mutation in the Btk gene had reduced neutrophil and monocyte recruitment to the peritoneum following zymosan challenge. To better understand the role of BTK in myeloid cell recruitment we investigated both chemotaxis and chemokine production in monocytes and macrophages. Pharmacological or genetic inhibition of BTK signalling substantially reduced human monocyte and murine macrophage chemotaxis to a range of chemoattractants (complement C5a and CCL2). We also demonstrated that inhibition of BTK in tissue resident macrophages significantly decreases chemokine secretion by reducing NF-kB activity and Akt signalling. Our work has identified a new role of BTK in regulating myeloid cell recruitment via two mechanisms, 1) reducing monocyte/macrophages’ ability to undergo chemotaxis, and 2) reducing chemokine secretion, via reduced NF-kB activity in tissue resident macrophages.
Background and Purpose Ca2+ influx via TRPV4 triggers Ca2+ release from the IP3-sensitive internal store to generate repetitive oscillations. While mitochondria are acknowledged regulators of IP3-mediated Ca2+ release, how TRPV4-mediated Ca2+ signals are regulated by mitochondria is unknown. We show that depolarised mitochondria switch TRPV4 signalling from relying on Ca2+-induced Ca2+ release at IP3 receptors, to being independent of Ca2+ influx and instead mediated by ATP release via pannexins. Experimental Approach TRPV4 evoked Ca2+ signals were individually examined in hundreds of cells in the endothelium of rat mesenteric resistance arteries using the indicator Cal520. Key ResultsTRPV4 activation with GSK1016790A(GSK) generated repetitive Ca2+ oscillations that required Ca2+ influx. However, when the mitochondrial membrane potential was depolarised, by the uncoupler CCCP or complex I inhibitor rotenone, TRPV4 activation generated large propagating, multicellular, Ca2+ waves in the absence of external Ca2+. The ATP synthase inhibitor oligomycin did not potentiate TRPV4 mediated Ca2+ signals. GSK-evoked Ca2+ waves, when mitochondria were depolarised, were blocked by the TRPV4 channel blocker HC067047, the SERCA inhibitor cyclopiazonic acid, the phospholipase C (PLC) blocker U73122 and the inositol triphosphate receptor (IP3 R) blocker caffeine. The Ca2+ waves were also inhibited by the extracellular ATP blockers suramin and apyrase and the pannexin blocker probenecid. Conclusion and Implications These results highlight a previously unknown role of mitochondria in shaping TRPV4 mediated Ca2+ signalling by facilitating ATP release. When mitochondria are depolarised, TRPV4-mediated release of ATP via pannexin channels activates plasma membrane purinergic receptors to trigger IP3 evoked Ca2+ release.
Since the start of the novel coronavirus SARS-Cov-2 pandemic, a disease that has become one of the world’s greatest global health challenges, the role of the immune system has been at the forefront of scientific studies. The pathophysiology of COVID-19 is complex, which is evident by those at higher risk for poor outcome. Multiple systems contribute to thrombosis and inflammation seen in COVID-19 patients, including neutrophil dysfunction, platelet activation, endothelial cell activation. Understanding how the immune system functions in different patient cohorts (particularly given recent emerging events with the Oxford/AstraZeneca vaccine) is vital to understanding the pathophysiology of this devastating disease and for subsequent development of novel therapeutic targets and expedite possible drug repurposing strategies that could benefit society on a global scale.
Pyroptosis, is a specialized form of inflammatory cell death which aids the defensive response against invading pathogens. Its tight regulation is lost during infection by the severe acute respiratory coronavirus 2 (SARS-CoV-2) and thus uncontrolled pyroptosis disrupts the immune system and the integrity of organs defining the critical conditions in patients with high viral load. Molecular pathways engaged downstream to the formation and stabilization of the inflammasome -required to execute the process- have been uncovered and drugs are available for their regulation. On the contrary, pharmacological inferring of the upstream events -which are critical to sense and interpret the initial damage by the pathogen- is far from being elucidated. This limits our capacity to identify early markers and targets to ameliorate SARS-CoV-2 linked pyroptosis. Here we aim to raise attention on mitochondria and pathways leading to its dysfunction with the goal to inform early steps of inflammasome and devise tools to interpret and counteract diseases by the SARS-CoV-2.